Reactors are used for a variety of physical, chemical and biological processing methods. Most reactors, including bioreactors, are complex mechanical devices that provide mainly the mixing and gasification of liquids to produce a variety of products. Bioreactors constitute a special type of reactors that are used to manufacture a variety of biological products ranging from wine to insulin (fermenters are a special type of bioreactors used for anaerobic reactions, even though the term fermenter is widely used to refer to bioreactors in general). The manufacturing of biological products involves several additional unit processes besides the process of growing the biological entities, all of which are tedious, expensive and cumbersome and completed outside the bioreactors, making the cost of manufacturing of biological drugs very high. The current methods of mixing and gasification used in bioreactors are also non-conducive to optimal production of biological products, further increasing their cost of manufacturing. There is a great unmet need for creating a reactor system that will be capable of producing biological products under the most optimal conditions, be able to combine all steps of biological product manufacturing within the same container and be of the lowest cost to own and operate. Such an invention will change the course of drug discovery and manufacturing, making it possible to provide life-saving new drugs to mankind at an affordable cost. Independently, the invention will serve many other industries where the it can be used for such specific purposes as mixing, gasification and sterilizing and holding solutions.
Liquid mixing is a major unit process in many industries including bioprocessing, chemical, and pharmaceutical manufacturing. The common methods of mixing a liquid (or a mixture of liquids and solids) in a container include use of an impeller, rocking or shaking the container, sparging gases to move liquid, ultrasonic waves and several combinations of these methods. Whereas, many of these mixing systems provide adequate quality of mixing as desired by the process, mixing of liquid in large containers, particularly the flexible disposable containers, remains a major problem. There is an unmet need to devise systems that will consume the least amount of energy, produce the lowest stress on the contents mixed, achieve mixing in the shortest period of time and produce consistent mixing results. Since mixing is a often an ancillary step in many other processes such a bioreaction, chemical reaction and manufacture of products, an efficient mixing system will yield highly profitable and time-saving operations in many industries. Whereas the term “liquid” is used in the present invention a fluid material, it refers to a nutrient medium or a culture medium or to a mixture of nutrient or culture medium and a biological culture interchangeably.
A variety of containers and methods have been developed over the years to carry out the mixing in the fermentation of microorganisms, particularly bacteria and yeast, on a commercial scale. Mixing in bioreactors serves many functions including diluting the metabolites, expel the gases produced and providing uniform gasification. The fermentation processes are generally conducted in stainless steel fermentation containers of several hundreds of thousands liters, with the fermentation methods including batch, fed-batch, continuous or semi-continuous perfusion. The cells within these containers are desirably kept in suspension, typically by rotating stirring blades located within the container, with gas exchange facilitated by the injection of air, oxygen or carbon dioxide into the container.
Gasification is a process of adding a specific gas to a nutrient medium; more commonly this includes adding gases like oxygen to grow biological organisms and tissues or using an inert gas to remove other gases like oxygen. A large number of applications from sewage treatment to bioprocessing of therapeutic proteins to operation of aquariums are dependent on gasification of liquids. In most hard-walled containers, gases are introduced by a sparger, a device with plurality of pores that diffuse gases inside a nutrient medium. A large variety of spargers are used, from the slow bubbling fish aquarium type to high-speed single point nozzles for the aeration of bioreactors. The efficiency of sparger is measured in the KLA value or liquid-gas transport coefficient that describes how fast a gas saturation is reached. It is expressed as liters per hour of gas that is absorbed in a liquid. For example, at room temperature (25° C.) the solubility of oxygen in water is about 8%; this decreases to about 6.5% at 37° C., the temperature most often used for bioprocessing. How fast is this maximum concentration of oxygen is reached in water upon starting gasification is a function of rate of gasification and mixing; also critical is the size of the bubble and thus the surface area of gas exposed to water. It is almost impossible to predict the KLA values since so many factors impact this value. However, higher efficiency systems of gasification allow for very high values of KLA, from 100-5000.
The above two processes form the critical requirement for a bioreactor design since the primary function is to provide sufficient gasification while mixing a nutrient medium in the presence of a biological culture. The current methods of fermentation have several drawbacks to their design. One is the introduction of shearing forces through the stirring blades and the cavitation of miniscule gas bubbles, both being detrimental to more sensitive cell types or organisms. Also, these containers should be rigorously cleaned between production runs to prevent cross-contamination, the latter being time consuming and requiring validation for individual cultures. Furthermore, the cost of stirred fermentors is relatively high on a volume basis, and thus these fermentors are commonly used over long periods of time. This, however, increases the risk of undesirable infection of mechanical failures. Perhaps most significantly, the optimization of culture conditions for stirred fermentors in a small scale cannot be transferred in a linear way to commercial scale production. For example, the fluid dynamics, aeration, foaming and cell growth properties change when the scale increases. In addition, for more delicate cell types or organisms, a large scale stirred fermentation container is not a viable device, even when more subtle stirring techniques such as airlift fermentors are used.
These drawbacks have led to the development of disposable fermentors. Examples of such disposable fermentors are systems based on wave agitation. See, e.g., U.S. Pat. No. 6,544,788; PCT Publication WO 00/66706. This type of fermentor may be used to culture relatively sensitive cells such as CHO cells (e.g., Pierce, Bioprocessing J. 3: 51-56 (2004)), hybridoma cells (e.g., Ling et al., Biotech. Prog., 19: 158-162 (2003)), insect cells (e.g., Weber et al., Cytotech. 38: 77-85 (2002)) and anchorage-dependent cells (e.g., Singh, Cytotech. 30: 149-158 (1999)) in a single disposable container. Such disposable units are relatively cheap, decrease the risk of infection because of their single use and require no internal stirring parts as the rocking platform upon which these containers reside during use induces wave-like forms in the internal nutrient medium which facilitates gas exchange. However, this principle cannot be expanded to the size of hundreds of thousands of liters (such as the industrial fermentors) but are currently available from 1 liter to 500 liters (total volume of the disposable bag, available from Wave Biotechnology AG, Switzerland; Wave Biotech Inc., USA). Moreover, the hydrodynamics for each size of disposable bag will differ as a result of differences in depth and height. Therefore, the use of these disposable bags requires optimization and re-validation of each step in an up-scaling process.
Nutrient medium mixing is a major unit process in many industries including bioprocessing, chemical, and pharmaceutical manufacturing. The common methods of mixing a nutrient medium (or a mixture of liquids and solids) in a container include use of an impeller, rocking or shaking the container, sparging gases to move liquid, ultrasonic waves and several combinations of these methods. Whereas, many of these mixing systems provide adequate quality of mixing as desired by the process, mixing of nutrient medium in large containers, particularly the flexible disposable containers, remains a major problem. There is an unmet need to devise systems that will consume least amount of energy, produce lowest on the contents mixed, achieve mixing in the shortest period of time and produce consistent mixing results. Since mixing is a often an ancillary step in many other processes such a bioreaction, chemical reaction and manufacture of products, an efficient mixing system will yield highly profitable and time-saving operations in many industries.
There remains an unmet need to develop a sparging system that will allow a uniform and quick dispersion of gases throughout the liquid, reducing dependence on mixing to achieve a uniform concentration. This is of greater use in the deployment of bioreactors.
While the bioreactors are exclusively used for the purpose of growing bacteria or other cells, their role can be expanded to include other processes that can be completed within the bioreactor. There is an unmet need to develop a bioreactor for expressing and separating a biological product from other components in the culture medium, combining the steps of expressing and separating within the bioreactor by binding the biological product with a resin within a bioreactor, discarding the nutrient medium and eluting the biological product as a concentrated solution; this will eliminate at least two steps in the separation and purification of biological products—filtration or centrifugation to remove cell culture and ultrafiltration for volume reduction—and possibly three steps, including loading of biological products on the purification columns. For products which are expressed as inclusion bodies, the present will involve lysing the cells, solubilizing the inclusion bodies, folding the protein prior to binding it to a resin, all within the bioreactor.
A combination bioreactor will be more appropriately called a preparative bioreactor and this will significantly reduce the process time and cost while enhancing the yield by reducing the degradation of biological products during manufacturing. No such invention exists in the prior art of bioreactors.
Downstream processing involves steps for cleaning up crude biological products to yield high purity products. Traditionally, these steps involve using chromatography columns packed with highly specialized resins to capture and purify the desired biological products by the process of elution. With an exponential rise in the number of biological products being developed and marketed, there have been remarkable developments in the field of downstream processing; these have however not caught up with the developments in the upstream processing. A few years ago, an yield of 0.25 G of biological product per liter expressed by CHO cells was considered very high; today, we are hovering yields around 10 G/L making it possible to accumulate a very large quantity of biological products, particularly as the sizes of bioreactors have increased to thousands of liters. There are three steps that connect the upstream and downstream processing. First, the culture media must be filtered using fine filters (e.g., 0.22 microns) to remove cells (CHO cells have average size of 5 microns). This step utilizes an array of filters since the cells are likely to choke the filter surface easily and also require installing containers that will receive the filtrate. This requires containers of thousands of liters of capacity to match the size of the bioreactors. The next step is the reduction of the volume of filtrate since it is not possible to load such large volumes on columns that have limited flow rate. This is the stage where most often a cross-flow type filtration is used, again with a large bank of filters to complete the concentration process as quickly as possible. The mechanism of cross flow filtration place severe pressure on the solution and causes breakdown and precipitation of biological products resulting in losses of generally 10-20% at this stage. Both of these processes take a very long time and during this processing it is not possible to keep the biological product solution at a lower temperature resulting in the degradation of biological product as well. The third step is to load the concentrated solution in a chromatography column containing a binding media, a specific resin with affinity for the target biological product. Even though the volume of nutrient medium has been reduced considerably at this stage, the loading steps, nevertheless, takes substantial time to complete the loading.
The time and cost-consuming steps of filtration, chromatography and purification slow down the manufacturing process and add substantial capital cost requirement to establish cGMP-grade manufacturing operations.
Bioreactors used in the upstream processing are generally containers that allow growth of cell culture to express biological products and for reasons historic and traditional, a clear demarcation line exists between the expression of biological product and its purification. For this reason, no innovations have been made to add additional functions to the design of bioreactors while they do provide a large investment in a container that could possibly have multiple uses.
There is a large unmet need to stream line the entire process of biological manufacturing of products where the cost of manufacturing can be reduced substantially but combining several traditional steps in a single container, the bioreactor. Although bioreactor systems and related processes are known, improvements to such systems and processes will be useful in the preparation of a variety of products produced from a biological source at a substantially reduced cost, time and labor.